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Mechanism of Transcription Through the Nucleosome by Eukaryotic RNA Polymerase

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Science  12 Dec 1997:
Vol. 278, Issue 5345, pp. 1960-1963
DOI: 10.1126/science.278.5345.1960

Abstract

Nucleosomes, the nucleohistone subunits of chromatin, are present on transcribed eukaryotic genes but do not prevent transcription. It is shown here that the large yeast RNA polymerase III transcribes through a single nucleosome. This takes place through a direct internal nucleosome transfer in which histones never leave the DNA template. During this process, the polymerase pauses with a pronounced periodicity of 10 to 11 base pairs, which is consistent with restricted rotation in the DNA loop formed during transfer. Transcription through nucleosomes by the eukaryotic enzyme and by much smaller prokaryotic RNA polymerases thus shares many features, reflecting an important property of nucleosomes.

Many transcribed genes are covered with nucleosomes (1, 2), which raises the question of how the polymerase negotiates its obstructed passage (3, 4). When the bacteriophage SP6 RNA polymerase transcribes through the nucleosome, the histone octamer steps around the polymerase by forming an intranucleosomal DNA loop (5). The looped intermediate causes intermittent pausing during the advance of the polymerase (6). Here we show that this mechanism is also relevant for eukaryotic RNA polymerases.

The ability of eukaryotic RNA polymerases to transcribe chromatin templates in vitro has been demonstrated (7-13), but the mechanism of transcription through the nucleosome remains obscure. Here we describe a new system for comparing transcription of identical nucleosomal templates by phage SP6 RNA polymerase and yeast RNA polymerase III (Pol III).

The template used for transcription was a 227–base pair (bp) Sac I–Nco I fragment (5) containing a positioned nucleosome and an SP6 promoter (Fig. 1A). DNA and nucleosomal templates labeled at the Nco I end were transcribed for different lengths of time (Fig. 1B). Elongation complexes were formed on DNA and nucleosomal templates with similar efficiency (on 25 to 30% of the templates). When elongation was continued for 5 min, the intensity of the low-mobility band was substantially reduced, which suggests that transcription on a majority of the templates was completed. Quantitative recovery of label from the elongation complexes in the nucleosomal band (naked DNA was not generated) suggested that nucleosomes survive transcription by Pol III.

Figure 1

Analysis of the fate of nucleosomes on transcription by Pol III. (A) The 227-bp Sac I–Nco I template. The principal positions of the nucleosome core before (bold oval) and after transcription (light oval) are indicated. Initiation sites for yeast Pol III and phage SP6 RNA polymerases are indicated, and sequences of RNA synthesized in the absence of GTP or CTP, respectively, are underlined. A 4-nt 3' overhang at the Sac I end promotes efficient end-directed initiation of transcription by yeast RNA Pol III (23). (B) Nucleosomes re-form after transcription by Pol III. Analysis of DNA and nucleosome cores labeled at the Nco I end in a nucleoprotein gel is shown (5). Mobilities of the 227-bp DNA, nucleosomes, and Pol III elongation complexes are indicated. Initiation and early elongation complexes were preformed in the absence of nucleotides (–NTP) or GTP (–G) and transcribed for 2 s or 5 min on naked (DNA) or nucleosomal (Nucl.) DNA, as indicated (24). As there is no G in the first 15 nt of the transcript, RNA chain-elongating Pol III advances to bp 15 and then arrests when provided only with CTP, ATP, and UTP. RNA chain elongation resumes promptly upon addition of GTP (25). M, Msp I digest of pBR322; NTP, nucleoside triphosphates; GTP, guanosine triphosphate. (C) Transcription by Pol III induces internal transfer of a nucleosome core. The initiation (–NTP) and early elongation (–GTP) complexes, as well as complexes transcribed for 5 min (+Tr.), were digested with a saturating amount of Hae II as indicated above each lane. (D) Direct, internal nucleosome transfer in the presence of an excess of competitor DNA. Nucleosome-bearing DNA was transcribed for 4 s in the absence, or for 5 min in the presence, of unlabeled competitor DNA (final concentrations are indicated in micrograms per milliliter). Nucleosome cores were formed and analyzed as described (5); they were pre-digested with Hae II restriction enzyme (26) and purified in preparative nucleoprotein gel (6). Yeast RNA Pol III was purified as described (27).

Does the position of the nucleosome core change during transcription? Positions of nucleosomes before and after transcription were analyzed with a restriction enzyme assay [Fig. 1C (5,11)]. Only ∼20% of nucleosomes were sensitive to digestion in the presence of Hae II restriction endonuclease before transcription, which indicates that most nucleosomes were positioned at the Nco I end. In contrast, the sensitivity of most nucleosome cores to this enzyme after transcription indicated quantitative transfer to the Sac I end. The amount of label in the free-DNA band did not increase after transfer. In contrast, ∼10% of nucleosomes fall apart on transcription by the SP6 polymerase (5), which suggests that Pol III–dependent transfer of the histone octamer is more efficient.

The mobility of a nucleosome in the gel indicates its position on a DNA fragment with ∼10-bp resolution; nucleosomes positioned symmetrically about the fragment center have the same mobility (14,15). Therefore, the very similar mobilities of nucleosomes before and after transcription indicate transfer from the Nco I end to the Sac I end of the fragment over a distance of ∼80 bp, which is very similar to the distance of transfer when SP6 polymerase transcribes the same template (5).

Is the histone octamer transferred within the same DNA molecule? Labeled nucleosomes were transcribed in the presence of excess unlabeled competitor DNA (Fig. 1D). The amount of free labeled DNA increased after transcription, indicating appreciable transfer of the histone octamer to unlabeled competitor DNA (5). However even in the presence of competitor DNA, 50 to 60% of the label from the elongation complexes was still recovered in the nucleosome band, which suggests relatively efficient nucleosome transfer by a direct mechanism within the same DNA molecule. In comparison, only ∼30% of nucleosomes survived transcription by SP6 polymerase in the presence of competitor DNA (5), again suggesting more efficient internal nucleosome transfer than with SP6 polymerase.

The pace of transcription of naked DNA and nucleosomes by Pol III and SP6 RNA polymerase was also analyzed (Fig.2). Both polymerases quickly completed transcripts on the naked DNA template. The broad size distribution of transcripts at early time points (2 and 4 s) has been analytically modeled in terms of the existence of two states of RNA polymerase at each step of RNA chain elongation: one competent to add the next nucleotide rapidly and the other not (16). Recent results suggest a structural interpretation of the elongation-blocked state: It arises when the RNA polymerase catalytic site moves out of register with the 3′ end of the nascent transcript (17-19); if the RNA-DNA duplex and transcription bubble-shift concomitantly, the growing point of the RNA chain disengages from the DNA template strand (17).

Figure 2

Analysis of nucleosome-specific pausing. (A) Time courses of transcript elongation by Pol III on the 227-bp template (28). Analysis of transcripts on denaturing polyacrylamide gel electrophoresis is shown. Cores and 227-bp DNA were transcribed at 20°C for different times (from left to right: 2, 4, 8, 30, and 180 s) after formation of early elongation complexes (–G). Samples were also incubated with ribonuclease (RNase) A (A) or RNase H (H) as indicated after 180 s of transcription. Transcripts were sensitive to RNase A and resistant to RNase H, indicating no formation of extended DNA-RNA hybrids. The original position of the nucleosome core is shown in the inset at the right: The region of strong nucleosome-induced pausing is indicated by a black box, the regions of less intense pausing by shaded boxes, and the regions of weak pausing by dashed lines; the nucleosomal dyad is indicated. SP6 polymerase runoff transcripts derived from a mixture of different restriction digests of pD70 were used as RNA markers (M) (6) (sizes are indicated on the left). (B) Time courses of transcript elongation by SP6 polymerase. Cores were transcribed for 4, 10, 25, 60, 180, and 600 s at 0°C after formation of early (14-oligomer) elongation complexes (–C). (C) Schematic diagram of nucleosome-induced pausing of the Pol III and SP6 RNA polymerases on the 227-bp template. The pausing patterns (after 8 s of transcription by Pol III or 25 s of transcription by SP6 polymerase) are plotted as black bars with heights directly proportional to the intensities of the corresponding bands in the gel. Transcription by SP6 RNA polymerase was as described (5, 6).

Nucleosomal templates slowed transcription, with SP6 and Pol III elongation complexes encountering obstruction predominantly within the promoter-proximal half of nucleosomal DNA. Nucleosome-generated obstructions to RNA chain elongation by Pol III were clustered, with an apparent periodicity of 10 to 11 nucleotides (nt) (Fig. 2C), generating peaks at about nt 105, 116, 126, and 137 after 4 s of RNA synthesis (Fig. 2A). Although some obstructions were transient (at nt 105 and 126), others persisted (at nt 116 and 137). Other transcripts (at nt 104, 140, 142, 147, and 150) were less identifiable with nucleosome-generated arrest; for example, the lack of an apparent precursor, and delayed appearance, of the nt 104 transcript points to events subsequent to obstruction of elongation. Although many transcripts were never completed during the allotted time (Fig. 2A), experiments with sarkosyl, which strips nucleosomes from DNA but leaves elongation complexes intact and able to complete nascent transcripts (20), indicated that at least 50% of obstructed polymerases were still functional at the last point of the time course (21).

The similarity of the Pol III– and SP6-specific pausing patterns strongly suggests that the same mechanism operates in both cases. This is surprising in view of the different sizes of the polymerases: Phage SP6 RNA polymerase is a small (∼100 kD) single-subunit prokaryotic enzyme whereas yeast Pol III is one of the large (∼600 kD), multisubunit, eukaryotic nuclear RNA polymerases. If, as previously proposed, the intranucleosomal DNA loop prevents rotation of either RNA polymerase around DNA, then it must be broken to allow continued elongation (6). When this occurs, even the much larger eukaryotic polymerase can continue RNA elongation until the loop forms again, forcing the enzyme to pause once more. Obstruction by the nucleosome can also drive elongating Pol III into a relatively prolonged state of blocked elongation.

The similar distances of the nucleosome transfers and the similarity of the SP6 polymerase and Pol III pausing patterns strongly suggest similar topographies of the transfer intermediates (Fig. 3). Why is the 10- to 11-bp periodicity of pausing prominent in the case of Pol III? It seems reasonable to propose that because of the much larger size of Pol III, the DNA loop only forms when the polymerase is positioned precisely on the outside of the loop; rotation of the Pol III molecule is almost completely restricted after the loop is formed. Because the rotational orientation of DNA is fixed by the remaining DNA-histone contacts, and if only one rotational orientation of the polymerase is favorable for formation of the loop, the transfer intermediates would be formed with the observed 10- to 11-bp periodicity. The much weaker periodicity in the case of SP6 polymerase may result from less severe restrictions on rotation of the smaller polymerase in the DNA loop. The smaller enzyme might be able to continue limited elongation (probably 2 to 5 nt) even after loop formation and might then stop only when it meets a DNA sequence intrinsically unfavorable for elongation.

Figure 3

A mechanism for transcription through a nucleosome. (A) RNA polymerase approaches the nucleosome. DNA in the elongation complex is severely bent (29); nucleosomal DNA is shaded. (B) A proposed structure of the transfer intermediate containing the intranucleosomal DNA loop. The relatively small size of the loop and the large size of Pol III result in transcriptional arrest with the observed 10-bp periodicity. In contrast, the much smaller SP6 RNA polymerase (right, in brackets) can continue to advance somewhat after loop formation. (C) Octamer transfer is completed.

Why is Pol III–induced transfer more efficient? The efficiency of SP6-dependent intramolecular nucleosome transfer is known to be greater at a lower elongation rate (5); the greater transfer efficiency by Pol III could be due to its intrinsically slower elongation rate.

What might be the consequences of direct transfer in vivo? Transcription would induce nucleosome translocation toward the promoter, depleting nucleosomes from the 3' end of the gene; of course, nucleosomes could be transferred back to this region (5). Thus, the nucleosomal organization of a transcribed gene should be dynamic (22).

Our results show clearly that a eukaryotic polymerase is capable of transcribing through a nucleosome without displacing it from the template. This ability reflects a property of nucleosomes that is likely to be of importance for the transcription process in vivo.

  • * Present address: Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, 540 East Canfield Avenue, Detroit, MI 48201, USA.

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